Infection with Human Immunodeficiency Virus type 1 (HIV-1) results in dysregulation and the ultimate depletion of the immune system known as Acquired Immunodeficiency Syndrome (AIDS). CD4+ T cells are gradually depleted and the infected individuals eventually succumb to a variety of opportunistic infections. The exact mechanisms of how HIV-1 induces AIDS are not well understood. It is clear that infection with HIV-1 results in depletion of CD4+ T cells and a state of hyperactivation of the immune system. However, the exact mechanism of these processes remains elusive. In this application, we propose to explore what role the viral protein Nef might play in AIDS pathogenesis. Our preliminary work shows that Nef is secreted from HIV infected cells in the form of exosome-like vesicles. Nef vesicles are present in the plasma of infected individuals and our data suggests Nef vesicle levels correlate with recovery of CD4+ T cells after antiretroviral therapy. We hypothesize that Nef, secreted from infected cells in the form of vesicles can induce effects on T cells and macrophage and cause at least some of the pathogenesis observed in AIDS. In this application we propose to study vesicular Nef and its interaction with primary T cells and macrophage. The composition of Nef vesicles will be determined using a combination of magnetic bead separation followed by characterization of bound vesicles by flow cytometry and Proteomics. Nef vesicles isolated from the plasma of infected individuals will be used to treat primary CD4 + and CD8 + T cells and macrophage. The treated cells will be analyzed to determine induction of apoptosis, immune activation, cytokine expression and gene expression using 96-well arrays. Together the information gathered will give us a better picture of the role of secreted Nef in the pathogenesis of AIDS. Such information could lead to new therapeutic approaches that target secretion or the action of secreted Nef. PUBLIC HEALTH RELEVANCE: HIV-AIDS is disease characterized by the destruction of normal immunity. In this application we propose that infection with HIV causes the release of large numbers of virus-like packets called "exosomes". Our theory is that release of exosomes is responsible for at least some of the loss of immune function seen in AIDS.